The invention relates to the field of biology, and more particularly to that of therapeutic chemistry.
The invention relates to pharmaceutical and/or dietary compositions based on an enzyme or an enzymatic complex comprising this enzyme, which can contribute towards prevention and/or treatment of diseases.
It more particularly relates to pharmaceutical compositions based on an enzyme or an enzymatic complex comprising this enzyme which stimulate the functions of the intestine and can thus contribute towards prevention and/or treatment of degenerative diseases associated with ageing.
It specifically relates to pharmaceutical compositions comprising, as the active principle, one or more enzymes extracted from or contained in the enzymatic complex obtained by culture of Streptomyces fradiae under the conditions defined in French Patent 2,600,340 and in the U.S. Pat. No. 5,047,240 issued Sep. 10, 1991, in the name of the Applicant.
The above-mentioned patent has described the production and use in zootechnology of a new proteolytic complex obtained from a filamentous bacterium, Streptomyces fradiae; in the following, this complex is called by its current commercial name: PANSTIMASE(copyright).
This filamentous bacterium can produce simultaneously at least 5 proteinases and 2 peptidases; these 7 enzymes have been described by K. MORIHARA et al. (Biochem. Biophys. Acta, 1967, 139, 382). When they are added together to foods consumed by animals, these 7 enzymes cause an excessive reduction of the viscosity of the intestinal mucus and can then cause ulceration of the intestinal wall. These 7 enzymes thus have adverse effects in zootechnology if they are used together.
Under the conditions indicated in the above-mentioned U.S. patent, PANSTIMASE(copyright), which is made up only of 3 exocellular proteinases, with a preponderance of one of them, and with the exclusion of endocellular proteinases and peptidases, is obtained. When added to foods consumed by animals, PANSTIMASE(copyright) causes a moderate reduction in the viscosity of the intestinal mucus, and it is not capable of attacking the intestinal wall.
At the same time as having a relatively moderate action on the proteins of the intestinal mucus, PANSTIMASE(copyright) has a more potent action on the proteins of the raw materials used in the composition of foods, which improves their digestibility.
In addition to these direct actions in the intestine, PANSTIMASE(copyright) has indirect actions on various organs, in particular on the pancreas; as already indicated in the earlier patent, it has been found that it causes an increase of 50 to 80% in the concentration of enzymes or proenzymes in this organ.
Because of these favourable actions, PANSTIMASE(copyright) is now used as a nutritional complement added to foods intended for animals to improve their growth or productivity. It has thus been found that it also improves the resistance of animals to infectious, bacterial or viral diseases, and that it reduces certain adverse effects of ageing.
These findings have suggested the possible use in therapeutics, for both animals and man, of PANSTIMASE(copyright) or of the active principles of which it is made up, that is to say the preponderant exocellular proteinase produced by Streptomyces fradiae or the mixture of this preponderant exocellular proteinase with at least one of the other two exocellular proteinases produced by Streptomyces fradiae. 
The active principles of pharmaceutical compositions require additional purification compared with the product used on animals. Under the conditions indicated in the earlier patent, non-purified PANSTIMASE(copyright) is obtained, the titre of which is at least equal to 2,000 A.U./mg.
The Anson Unit (abbreviated to A.U.) is defined here as being the amount of enzyme which, when incubated for 10 min at 25xc2x0 C. and at pH 7.5 in the presence of denatured haemoglobin, liberates from this substrate the equivalent of 1 mcg tyrosine, determined by spectrophotometric absorption at 280 nm on the filtrate which cannot be precipitated by trichloroacetic acid.
Various adjustments to the conditions indicated in the earlier patent allow a semi-purified PANSTIMASE(copyright) to be obtained, the titre of which is at least equal to 4,000 A.U./mg. The conventional techniques of fractional precipitation or passage over a column enable each of the 3 proteins of which PANSTIMASE(copyright) is composed to be obtained in isolation. The specific activity of the preponderant exocellular proteinase is greater than 100,000 A.U./mg, and is significantly higher than that of the other 2 proteinases; its molecular weight is about 19 kDa and its isoelectric point is about 8.5.
This preponderant proteinase alone explains the main physiological properties of PANSTIMASE(copyright), in particular because of the fact that it alone has a characteristic action on two important proteins: cholera toxin and collagen.
Cholera toxin is the homologue of LT toxin produced by some pathogenic colibacilli; it is composed of the sub-unit A, which is aggressive, and the sub-unit B, which is immunostimulating. At a low concentration comparable to that which exists in the intestine of animals receiving food supplemented with PANSTIMASE(copyright), the preponderant proteinase selectively destroys the sub-unit A without destroying the sub-unit B, which therefore keeps its capacity to stimulate the immune system.
Collagen is the most abundant protein in the animal kingdom: in mammals, the various types of collagen represent about one third of the total proteins. It is known that the solubility of muscular collagen in hot water is high if it originates from young animals and low if it originates from elderly animals.
When the collagen of elderly animals is treated with the preponderant proteinase, its solubility in hot water increases and becomes comparable to that for young animals. It can be concluded that this proteinase causes a certain xe2x80x9crejuvenationxe2x80x9d of collagen. The majority of results on animals correspond to tests carried out with non-purified or semi-purified PANSTIMASE(copyright). For man, it could be possible to use either semi-purified PANSTIMASE(copyright) or the purified proteinase or proteinases of which it is composed, that is to say the preponderant exocellular proteinase or the mixture of this preponderant exocellular proteinase with at least one of the other two exocellular proteinases, or with a proteinase or proteinases of which the amino acid sequences have at least 60% homology with those of the preponderant exocellular proteinase or with those of the mixture of proteinases as defined above.
The present invention relates to pharmaceutical and/or dietary compositions comprising, as the active principle, the preponderant exocellular proteinase produced by culture of Streptomyces fradiae, which has a specific activity greater than 100,000 A.U/mg, a molecular weight around 19 kDa, and an isoelectric point around 8.5, and is capable of selectivity destroying the sub-unit A of cholera toxin without destroying the sub-unit B, as a mixture or in combination with an inert, nontoxic, physiologically acceptable excipient or vehicle.
The present invention also relates to pharmaceutical and/or dietary compositions comprising, as the active principle, the preponderant exocellular proteinase as defined, or the mixture of this preponderant exocellular proteinase with at least one of the other two exocellular proteinases produced by culture of Streptomyces fradiae, or a proteinase or proteinases of which the amino acid sequences have at least 60% homology with those of the preponderant exocellular proteinase or with those of the mixture of proteinases as defined above, as a mixture or in combination with an inert, non-toxic, physiological acceptable excipient or vehicle. The homologous proteinases can be extracts of the culture of various microorganisms, and in particular Streptomyces fradiae or Streptomyces griseus. 
The active principle is administered by the oral, pulmonary or genital route in doses of between 5,000 and 100,000 A.U. For this purpose, the active principle, as a mixture or in combination with conventional excipients, if brought into one of the pharmaceutical form suitable for these types of administration.
The active principle is present in particular in one of the customary forms in pharmacotechnology, such as tablets, coated tablets, gelatine capsules, capsules, powders, solutions or suspensions, dry aerosols, ovules and vaginal tablets.
The administration will be one to three times daily, depending on the therapeutic requirement.